Fmoc-POAC: [(9-Fluorenylmethyloxycarbonyl)-2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic Acid]: A Novel Protected Spin Labeled .BETA.-Amino Acid for Peptide and Protein Chemistry.

Abstract

TOAC,1) the stable free radical 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid,2) acylated at the amine function with the tert-butyloxycarbonyl group (Boc-TOAC), was the first protected spin-labeled amino acid derivative successfully introduced 3,4) for labeling peptides through the solid phase method. 5,6) However, due to partial decomposition 3,7) of the nitroxide moiety during the repeated trifluoroacetic acid (TFA) treatments necessary for Boc group 5) removal, labeling with this paramagnetic marker was restricted to the N-terminal position of the sequence. To overcome this shortcoming, the 9-fluorenylmethyloxycarbonyl (Fmoc) group 6) was used for protection of the amine function. Moreover, the conjugation of both Fmoc (for peptide chain elongation) and Boc (for peptide cleavage from the resin) chemistries allowed us to propose a procedure for TOAC insertion at any internal position of a peptide sequence. 8)Since its use for the synthesis of spin labeled peptides was first described, this C a -tetra-substituted cyclic a-amino acid was employed in a great variety of investigations. For instance, the first example of a labeled peptide hormone (amelanocyte stimulating hormone) that retained full biological activity was recently reported.9) Furthermore, a large sampling of mono-or doubly-labeled model peptides have been studied in terms of structural features. [10][11][12][13][14] As a further development in our research on spin labeled amino acids, here we describe the convenience of using the b-amino acid derivative 2,2,5,5-tetramethylpyrrolidine-Noxyl-3-amino-4-carboxylic acid (POAC), protected with the Fmoc group, for peptide labeling. Besides the inherent interest in using this alternative spin label for a broad range of chemical and biochemical purposes, an additional objective for introducing POAC in peptide chemistry is to overcome the serious difficulty in coupling the subsequent amino acid residue after TOAC incorporation.8) This is due to the very low nucleophilicity of its amine function when bound to a peptide sequence (pK a Ͻ6) 3,4) which can be overcome when a more efficient coupling procedure is applied. 8) In contrast, the amine function linked to the POAC pyrrolidine b-carbon should confer higher nucleophilicity than that in TOAC, facilitating the coupling reaction during peptide synthesis. The steps for POAC (3) synthesis are shown in Fig. 1 and have been described in detail elsewhere. 2,15) Owing to the presence of two asymmetric carbons in POAC's b-cyclic structure, its synthesis yields a racemic mixture. Crystals suitable for X-ray diffraction were crystals obtained by slow evaporation from an ethanol-water solution. Single crystal structure determination showed that a trans The stable free radical 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) is the only spin labeled amino acid that has been used to date to successfully label peptide sequences for structural studies.

Peptide–Membrane Interactions by Spin-Labeling EPR

“…Balog et al, 2003; M. R. Balog et al, 2004; Kalai, Schindler, Balog, Fogassy, & Hideg, 2008; Tominaga et al, 2001; Wright et al, 2007). The most commonly used one is 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid (TOAC, Figure 1, 8 ) (Rassat & Rey, 1967; Schreier, Bozelli, Marín, Vieira, & Nakaie, 2012; A.…”

Application of Electron Paramagnetic Resonance Spectroscopy for Validation of the Novel (AN+DN) Solvent Polarity Scale

“…Spin labels (A) and (B) are precursors of the amino acid-type spin probes 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-amino-4-carboxylic acid (POAC) [21,22], both further chemically derived for allowing their pioneering insertion within a peptide sequence [23][24][25]. The EPR spectra of these three spin probes were recorded for 15 single solvents, and binary correlations were drawn between the a N values and the (AN+DN) terms.…”

Synthesis of Enantiomerically Pure cis‐ and trans‐4‐Amino‐1‐oxyl‐2,2,6,6‐tetramethylpiperidine‐3‐carboxylic Acid: A Spin‐Labelled, Cyclic, Chiral β‐Amino Acid, and 3D‐Structural Analysis of a Doubly Spin‐Labelled β‐Hexapeptide

“…as its easy inclusion in a peptide synthesised by solid-phase methods had been demonstrated, [13] whereas similar syntheses with the TOAC residue proved problematic. [13,14] A β-amino acid form of TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-3-carboxylic acid, β-TOAC) was designed that could be obtained in enantiopure form.…”

“…[13,14] A β-amino acid form of TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-3-carboxylic acid, β-TOAC) was designed that could be obtained in enantiopure form. [15,16] The β-amino acids have been synthesised and studied in the last ten years since it was demonstrated that their oligomers may fold into helical conformations (stable in organic and aqueous solvents) and are resistant to enzymatic hydrolysis.…”